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Electorophysiological nature of the respiratory rhythm generation were studied using in vitro rat brainstem-spinal cord preparation from newborn rats. Rhythm generation-related neurons were identified in rostral ventrolateral medulla by analyzing the reset of the rhythm under various calcium concentrations. It was found that the experimental condition of calcium 0.2 mM and magnesium 5 mM, a condition that has been employed for blocking the synaptic transmission of the in vitro CNS ( Central Nervous System ) preparation, was not sufficient for the complete block of the synaptic transmission. Under this condition the intracellularly recorded synaptic potentials evoked by a single shock stimulations to afferent neurons were decreased, but large synaptic potentials could still be evoked after repetitive stimulations of afferent neurons, and even action potentials could often be evoked in thepostsynaptic neurons. Therefore, it is possible that rhythmic activities of neurons recorded under this condition may involve synaptic activities of neurons, and the presence of rhythm cannot be a conclusive evidence that pacemaker cells exist in the respiratory rhythm generation systems. The effects on the rhythm of antagonists of glutamate, a major excitatory transmitter in the CNS, was also studied. When glutamte antagonists, CNQX, APV or kynurenic acid, were applied, they dramatically decreased the rate of the rhythm. This is rather unexpected if the simple pacemaker hypothesis for the rhythmgeneration holds true. In conclusion, the obtained results suggest that the simplest hypothesis for the respiratory rhythm generation, a pacemaker cell hypothesis, has to be critically re-evaluated and a possible active involvement of excitatory transmitters in the rhythm generation has to be reconsidered.